The present invention is directed to chicken virus isolates or strains (Texas RB 3, Texas RB 4, HBS, F57-7, W/L 39, GAR 1) believed to be a new strain or strains of infectious bursal disease virus (IBDV) which infects the proventriculus, a vaccine made from these isolates, a method of treatment, vaccination scheme, and the like.
In accordance with the present invention, numerous experiments were conducted to determine the role of infectious bursal disease (IBD) virus in the induction of lesions associated with proventriculitis syndrome in chickens. Parameters examined included age of the chicken at IBD virus exposure, concentration of IBD virus at exposure the strain of IBD virus, dietary influence in the presence of IBD virus, mixed IBD virus infection, autoimmune mediated IBD reactions associated with lesion production, viral induced apoptotic tissue injury and isolation and characterization of the causative agent.
The experiments were carried out in SPF white leghorns, with the experimental birds being examined for the presence of gross and microscopic lesion at 4 and 11 days post challenge. Tissue homogenates were analyzed for the presence of IBDV at 4 and 11 days post challenge with Antigen Capture ELISA (AC-ELISA). Determinations of neutralizing antibody levels and IgG antibody responses were monitored as well as depletion of serum complement following IBDV infection. Physical parameters were also considered utilizing body weights and organ to body weight ratios to determine IBDV effects following experimental challenge.
Physical parameters indicated that the primary viral response is predominantly in the bursa of Fabricius, but changes were also noted in the proventriculus. Physical changes in the proventriculus occurred primarily during the acute stage of the IBD virus infection.
Gross and microscopic lesions in the proventriculus were exacerbated by the presence of copper sulfate supplementation in the feed. There were also very evident negative effects on the weight gain of SPF white leghorn chickens given copper sulfate feed supplementation.
Infectious bursal disease virus strains show different affinities for producing lesions in the proventriculus following challenge. Standard challenge IBD virus USDA/STC stain produced the most consistent lesions in the proventriculus often accompanied by hemorrhage. This correlates well with antibody enhanced pathology as observed with antigen-antibody complex activation of serum complement. Evidence of yet another mechanism of IBDV induced pathology, was demonstrated through the use of TUNEL apoptosis staining of infected tissues. The degree of apoptotic involvement was also reflected in the pathogenicity of the IBD virus strain used for challenge.
Infectious bursal disease viral inclusions were demonstrated by thin section electron microscopy in the proventriculus at 4 days post challenge. Viral inclusions resemble previously documented inclusions produced in the bursa following IBD infection.
In accordance with the present invention, infectious bursal disease viruses were isolated from broiler chickens experiencing proventriculitis in, for example, Oklahoma, Texas, West Virginia, and California.
IBDV Antigen Capture (AC) Elisa tested positive for Texas RB 3, Texas RB 4, HBS, F57-7, W/L 39, and GAR 1. Virus isolation in SPF embryonated eggs was completed and 3rd and final passage for the isolates. SPF chickens were inoculated with egg harvest for the isolates. Tissues from inoculated birds and histopathology confirmed lesions. AC-ELISA of tissue and serological confirmation of the isolates was performed.
Proventriculitis is a major problem to the broiler industry. These viruses may be one of the major causes of this condition and if this proves to be true, could be utilized as or in a vaccine to prevent the disease condition. The viruses are believed to be a significant finding in the search for a causative agent for proventriculitis in broiler chickens and as such may be utilized in the development of a vaccine or vaccines. The viruses can be attenuated to be used as a modified live vaccine or utilized in an inactivated form in a killed vaccine.
INTRODUCTION
BRIEF DESCRIPTION OF THE DRAWINGS
I. SECTION 1
DETERMINATION OF THE ROLE OF INFECTIOUS BURSAL DISEASE VIRUS STRAIN VARIANT 1084-E IN THE INDUCTION OF PROVENTRICULITIS IN SPF CHICKENS: AGE RELATED EFFECTS 
II. SECTION 2
DETERMINATION OF THE ROLE OF INFECTIOUS BURSAL DISEASE VIRUS STRAINS USDA/STC AND VARIANT 1084-E ON THE INDUCTION OF LESIONS ASSOCIATED WITH PROVENTRICULITIS: INFLUENCE OF VIRUS CONCENTRATION AND TIME 
III. SECTION 3
THE INTERACTION OF INFECTIOUS BURSAL DISEASE VIRUS, REO VIRUS, AND COPPER SULFATE IN THE PRODUCTION OF LESIONS ASSOCIATED WITH PROVENTRICULITIS IN SPF WHITE LEGHORN CHICKENS 
IV. SECTION 4
INFECTIOUS BURSAL DISEASE VIRUS INDUCED APOPTOSIS ASSOCIATED WITH PRODUCTION OF LESIONS IN THE PROVENTRICULUS AND BURSA 
V. SECTION 5
MEASUREMENT OF HEMOLYTIC COMPLEMENT LEVELS AND VIRUS NEUTRALIZING ANTIBODY TITERS 4, 6, 8 AND 11 DAYS POST CHALLENGE WITH INFECTIOUS BURSAL DISEASE VIRUS STRAINS USDA/STC, VARIANT E/DEL, AND VARIANT E/1084
VI. SECTION 6
THE INTERACTION OF INFECTIOUS BURSAL DISEASE VIRUS (USDA/STC) AND COPPER SULFATE IN THE PRODUCTION OF LESIONS ASSOCIATED WITH PROVENTRICULITIS IN BROILER CHICKENS 
VII. SECTION 7
ISOLATION AND CHARACTERIZATION OF INFECTIOUS BURSAL DISEASE VIRUS FROM BROILER CHICKENS EXHIBITING SYMPTOMS OF PROVENTRICULITIS 
VIII. FURTHER DISCUSSION
IX. APPENDIX CLAIMS ABSTRACT OF THE DISCLOSURE
The primary impetus for this invention came as a result of a request for help from a number of commercial poultry processors. Proventriculitis has been described in broiler chickens from a number of geographical locations. This syndrome is characterized by the enlargement of the proventriculus, with feed impaction, and structural weakness. Affected birds can be either normal or underweight at processing, with either a high or normal feed conversion efficiency. Problems generally manifest at the processing plant with breakage of the proventriculus during mechanical evisceration, resulting in an increased number of washouts, slowed or stopped processing lines, excessive trims, and higher than normal levels of condemnation. This proventriculitis syndrome has been linked to a number of environmental, nutritional, genetic, and infectious causes.
Preliminary studies indicate that infectious bursal disease virus is capable of producing lesions in the proventricular mucosa. While proventriculitis induced by IBDV alone does not exactly mimic what is observed in field situations, it is contented that IBDV plays the role of a facilitator, and thus presents an opportunity for other viral, bacterial, fungal or chemical pathogens to exert an effect.
Some objects of this invention are to: (1) attempt to establish infectious bursal disease virus as a causative agent in the induction of lesions associated with proventriculitis, (2) determine the pathogenic mechanism by which IBDV exerts an effect on the proventriculus, and (3) to isolate and characterize IBDV virus from field cases of proventriculitis.
Infectious bursal disease (IBD) is an acute viral infection which predominantly affects domestic chickens. This viral disease has also been described in commercial turkeys (22, 60, 81, 82, 119), and represents an occasional problem in ducks (81). Infectious bursal disease virus infection and replication occurs primarily in lymphoid tissues throughout the body. This results in induction of significant pathological lesions within the follicles of the bursa of Fabricius, which contains the highest concentration of lymphoid cells. When infection occurs at an early age ( less than 3 weeks), the disease syndrome may be further complicated by an accompanying immunosuppression (33, 76, 95).
In 1962, Cosgrove (20) first described this disease syndrome and an associated nephritis in a chicken flock in Gumboro, Delaware. Early studies gave conflicting information concerning this disease. It was later determined that the nephritis syndrome described was actually a variant strain of infectious bronchitis virus and another, as yet, unknown infectious agent (12). Studies of tissues harvested from infected birds revealed the presence of virus particles that were structurally different from infectious bronchitis virus. The name Gumboro disease was adopted to describe this new infectious disease syndrome (10,95).
Hitchner (50) proposed the final name of infectious bursal disease virus to identify the etiological agent responsible for inducing pathognomonic lesions of the cloacal bursa. It was finally characterized as a member of the Birnaviridae family, which reflects that it has a genome that consists of two segments of dsRNA (10,26,76).
Infectious bursal disease virus (IBDV) infection represents a disease of significant economic importance within the poultry industry. Economic impact of IBDV infection is manifested by the mortality it induces during the initial acute infection, and secondly from the induction of an immunosuppressive syndrome in chicks infected immediately after hatching. The virus is considered ubiquitous in nature, with a worldwide distribution in all major poultry producing areas with the exception of a few isolated islands in the south Pacific (76,95,119).
Vaccination procedures for this virus are practiced extensively by commercial producers and are such that all chickens become seropositive to IBDV during their growout (35). Successful vaccination programs are evident in that clinical cases of IBDV are extremely rare. However, IBDV infections do still occur, and usually become apparent only after the introduction of a secondary pathogen. Modifications of the IBDV infection can occur in the presence of maternal antibodies (135) or as a result of variant IBDV strains that induce immunosuppression with no obvious clinical disease (95,119).
Two serotypes of IBDV are currently recognized, they are designated as serotype 1 and serotype 2 (81). The serotype 1 IBDV group can be further divided into two subtypes, the so called xe2x80x9cClassicxe2x80x9d and xe2x80x9cVariantxe2x80x9d (76) . There is additional evidence of IBDV isolates that represent a viral group that is transitional between classic and variant (6,7,128). Serotype 1 IBDV classic viruses were the first to be described in chickens, and as such, there is a great deal of information available concerning their characteristics. Variant IBDV strains of serotype 1 represent a new faction of this viral disease syndrome. While variant strains of serotype 1 IBDV maintain structural similarities and replication strategies, they are capable of breaking through maternal antibody generated against classical subtypes (110, 112). The appearance of variants within the viral population appears to be a reflection of the intensity of the vaccination program, with most variants representing a viral subpopulation selected by the use of live IBDV vaccines. Documented variant strains found within serotype 1 IBDV show distinct sequence differences in the VP2 gene from the classic viruses, indicating that mutations of the viral genome also influence the emergence of variant IBDV strains (128).
Serotype 2 IBDV antibodies are found in both chickens and turkeys, suggesting that this IBDV serotype also has a widespread geographical distribution. This particular subtype is noted in particular for their inability to induce detectable clinical symptoms. The so-called apathogenesis of IBDV serotype 2 has been attributed to the virus lacking a selective tropism for the bursa of Fabricius (22,99). Recent studies have also linked this apathogenesis to the inability of serotype 2 IBDV to utilize macrophages for distribution of virus particles throughout the host. This observation was made utilizing in-vitro cell culture in which serotype 2 virus infected cells underwent a rapid lytic cycle (138). Presence of antibodies to serotype 2 IBDV does not confer protection against serotype 1 IBDV, although it has been determined that both serotypes share a high degree of genetic homology (43,59,60).
IBDV is a classified as a member of the Birnaviridae family, which has only one genus Birnavirus. The recognized prototype virus in this classification is infectious pancreatic necrosis virus (IPNV) of fish (trout). This virus family also includes Tellina virus (bivalve mollusks, oysters)(26), Drosophila X (fruit fly) (13), and Eel virus (fresh/saltwater eels). Cross virus neutralization tests indicate that all the aquatic viruses within this family are related to each other, but are different from the avian and insect isolates (26).
Infectious bursal disease virus particles range in size from 55-60 nm, possess an external single shell, are non enveloped, and exhibit icosahedral symmetry (26,39,48,68,101,104,119). The capsid consists of 32 capsomeres arranged in a skewed (dextro) 5:3:2 symmetry (47,101). The G+C content is 55.3%, with a purine to pyrimidine ratio of approximately 1. IBDV is resistant to inactivation by ether, chloroform, pH levels between 2.0 and 12.0, and has a Tm of 95.5xc2x0 C., (12).
The buoyant density of complete particles was determined by the use of cesium chloride gradients and ranges from 1.31 to 1.34 g/ml (47). Viral particles on the lower density range contain significantly lower levels of dsRNA or are completely empty (85). Lower density particles exhibit identical morphology but lack the proteins of the proper size and concentration to initiate an effective infection. Incomplete particles, are unable to initiate infection, and constitute approximately 10% of any given Birnavirus preparation (7,47,85,89).
The IBDV genome of serotype 1 consists of two segments of dsRNA (12, 88) of approximately equal size, segment A has a molecular weight of 3.4xc3x97106 and segment B has a molecular weight of 2.8xc3x97106 (10, 12 ,26, 47, 86, 95, 104, 119). Serotype 2 IBDV exhibit a difference in the size of both segment A and B which are smaller by approximately 70 bp and 20 bp respectively (10, 60), although there appears to be no difference in function (11, 98). Five viral proteins have been identified for IBDV; they are as follows with their approximate molecularweights: VP1(90,000), VP2 (44,000), VP3 (35,000), VP4 (28,000), and VP5 (17,000) (25,119). Segment A codes for a polyprotein within a single open reading frame (ORF) in the following order N-VP2-VP5-VP4-VP3-C (4, 63, 83, 117). Segment B codes for a single protein VP1 (83, 87, 103).
VP2 and VP3 are the major structural proteins (99, 130) and collectively constitute 91% of the total viral protein. VP1 and VP4 are considered minor proteins (enzymes) and VP5 of unknown function collectively represent the remaining 9% of viral protein. Viral protein one (VP1) codes for a viral RNA polymerase, which is a required element for replication of dsRNA. Since all IBDV""s encode their own viral polymerase, transcription and replication are initiated immediately after the virus penetrates the host cell without need for uncoating or any type of degradation of the nucleocapsid (131). Infectious bursal disease virus utilizes the strand displacement model of replication, with VP1 also functioning as a viral replicase (130). Viral protein four (VP4) codes for a viral protease which processes the large segment A polyprotein precursor into mature viral proteins (62,63,83,98). VP5 has no known function at this time (119).
Viral protein two (VP2) represents the major host protective immunogen (61) and functions in virus adsorption to the host cell (68). Variability in the VP2 amino acid sequence is found predominantly within the antigenic binding epitope. Significant changes in this genomic region are often associated with emergence of variant IBDV strains (3, 4, 11, 14, 31, 69, 98).
Neutralizing antibody from the host animal is generated against the antigenic structure within the VP2 protein (30). Development of neutralizing antibodies in the host animal against viral VP2 is highly conformational dependent (11). Experimental studies utilizing renatured VP2 protein demonstrated the crucial importance of maintaining the native state of the VP2 antigen binding epitope. The renatured VP2 was capable of binding neutralizing antibodies, however, antibodies generated against this protein were not neutralizing (98).
Viral protein three (VP3) contains two conformational independent non-overlapping binding epitopes (61, 127); one of these epitopes is conserved across serotypes and one is distinct for each serotype (87, 99). Antibodies specific to VP3 are the first to appear in detectable levels in chickens following infection (61).
Viral protein one (VP1) forms a dsRNA protein complex, by covalently binding knoblike proteinaceous structures to the ends of the two genomic segments, forming a circularized ring structure. Viral protein one (VP1) exhibits the characteristics of an RNA dependent RNA polymerase and functions also as an assembly protein. Genome linked proteins are common in Birnaviruses (87, 103, 131), and their presence Indicates that replication of nucleic acid is by strand displacement (semi-conservative) mechanism (13, 131) The physiological function of VP1 protein is still unknown, although it has indications of involvement in RNA replication as well as in virus assembly (83, 87).
Chickens infected with IBDV exhibit trembling and ruffled feathers with additional signs of depression and anorexia. These symptoms are accompanied by a profuse uratecontaining diarrhea with some indication of bile stasis. The diarrhea syndrome is further complicated by the birds refusal to eat or drink, with most infected birds exhibiting evidence of dehydration. During acute IBDV infection there is an initial elevation of body temperature, which falls below normal shortly before death. Blood calcium levels are lowered (20) and there is evidence of the presence of additional blood clotting deficiencies (122,124).
Clinical IBDV is most commonly recognized in susceptible 3 to 6 week-old chickens. Although an early age resistance to infection, regardless of maternal antibody has been proposed (29, 50, 100, 141). Infectious bursal disease virus infection, within the first week of hatch leads to severe defects of humoral immune response (49). Mortality from IBDV infection was shown to be associated with a severe depletion of complement (123, 124), and clotting abnormalities without changes in complement (124).
Virus persistence was measured in a number of studies which considered the amount, and the route of virus inoculation, as well as the age of the bird when the virus challenge was given. Virus could be visualized, using the electron microscope, within fours hours PI in the cecum, and within 5 hours in the duodenum and jejunum. It was determined that IBDV could be reisolated from infected organs within 24 hours post infection (PI), regardless of the age of the bird, the dose given, or the route in which it was administered. Virus could not be recovered beyond day 11, with birds challenged at 1, 7, and 14 days of age. Birds challenged with IBDV at 21 days of age tested positive for only 8 days (79). In general, older birds at challenge experience shorter periods of viremia, but have a more rapid and higher intensity immune response (141). Bayyari et al. described extended periods of viremia associated with increased levels of defective IBDV viral particles following attenuation of IBDV in cell culture (7).
Infectious bursal disease virus can be propagated in specific pathogen free (SPF) embryonated eggs on the chorioallantoic membrane (CAM) or by the yolk sac (YS) route at day 9 of embryonation. Chorioallantoic membrane inoculation is considered to be the most sensitive method of virus isolation and propagation. Embryos that become infected with IBDV show petechial hemorrhages on the head, trunk, and feet, as well as edema within the abdominal cavity. Internally, the livers of affected embryos may demonstrate necrosis, but more commonly are pale and take on a parboiled or cooked texture. The spleen is usually normal in size with the exception of variant IBDV where there is significant enlargement of the spleen, in both cases the spleen is generally pale and may occasionally demonstrate necrosis. Embryonic bursae are extremely small with occasional hemorrhage. Mortality, if it occurs, is usually between 3 to 5 days PI, and, as a general rule, is absent in the presence of variant IBDV strains. The highest concentrations of the IBDV virus are found in the CAM, body and viscera of the infected embryo (109).
Cell culture has become an increasingly economical and efficient tool for measuring virus effects outside (in-vitro) the host animal. A number of years ago it was determined that IBDV could be adapted to grow in cell culture. Infectious bursal disease virus propagation in primary cells derived from SPF chickens and/or embryos results in the highest yield of virus particles (70). Growth of IBDV, both classic and variant strains, has been accomplished in the following primary cells: chicken bursal lymphoid (CBL), chicken embryo kidney(CEK) and chicken embryo fibroblast (CEF)(18, 46, 75, 82, 96, 126). Viral proteins of most IBDV are present at detectable levels by 90 minutes post inoculation (PI) in CEF cells. Virus replication then enters a latent stage at approximately 4 hours (56), with mature virus particles evident as early as 6 hours PI (85), and mature virus particle release between 10-16 hours PI (59, 75, 77, 90, 94). Virus replication in CBL cells closely parallels that of CEF cells, however, IBDV infection of CEK cells is of extended duration (59). Cytopathic effects induced by IBDV infection are visible by 16 hours PI (75, 94) and virus titers generally peak at 48 hours PI (18). In addition, it has been determined that IBDV infection activates complement in cultured lymphoid cells (71), and that IBDV progeny virus release is followed within 2-8 hours by an increase in the synthesis of interferon (34).
Several mammalian cell lines RK-13, VERO, BGM-70, and MA-104 will also support IBDV growth (34,42,56,75,104). Virus particle yields are lower, and the replication cycle is prolonged when compared to IBDV propagation in chick primary cells (56,75). Repeated passage in these cell lines leads to loss of pathogenicity and represent a means by which highly pathogenic IBDV can be attenuated for utilization as vaccines (6,40,42,143).
Cell culture propagation of IBDV under conditions where there is a high concentration of virus particles (MOI) and limited cellular substrate will result in the production of incomplete virus particles. Upon further passage in cell culture virus stocks containing these defective interfering (DI) particles show reduced virus yields and reduced pathogenicity (6,7,84). The mechanism that triggers some IBDV viruses to produce DI particles and other IBDV not to, is not clear at this point in time (6,89).
Cytopathic effects (CPE) of IBDV infection in cell culture are described as plaque formation. Extensive passage in cell culture can induce IBDV to form plaques of various sizes. It has been determined that IBDV virus stocks which produce large plaque CPE are more virulent than those that produce small plaque CPE (23,73,113).
Cell culture propagation of IBDV is not without controversy. A number of researchers have reported conflicting results from cell culture derived IBDV stocks, especially when they are introduced back into the chicken. These differences have raised some concern as to whether in-vitro observations of IBDV infection correspond in any way to what happens in-vivo , although the use of in-vitro methods continues to be a widely accepted method of analysis for IBDV infection (54).
The presence of IBDV within infected tissues and/or the presence of antibodies following infection can be determined with a number of different laboratory tests. The agar gel precipitin test (AGPT) can be used to detect IBDV group specific antibody. The test can be both quantitative and qualitative, but is of extremely low sensitivity, and therefore misses detection of low virus concentrations and is only useful during the first few days of IBDV infection (58,109).
Virus neutralization assays are highly specific for the identification and characterization of IBDV isolates. The test is performed utilizing primary CEF cell monolayers. Infectious bursal disease virus of a known serotype is reacted at a constant concentration with varying amounts of immune serum. The antigen-antibody mixture is then inoculated onto cell monolayers and incubated. Following incubation the cells are fixed and stained. Endpoints are determined visually and neutralization index titers are expressed as the reciprocal of the highest dilution of antiserum that prevents IBDV from inducing cytopathic effect in cell culture monolayers. Infectious bursal disease viruses can be pathotyped using cross neutralization assays, and also quantitated (7,32,109).
Fluorescently labeled antibodies are now readily available, and offer a unique, yet diverse diagnostic tool. Immunofluorescent staining of enzyme treated formalin fixed tissues using IBDV polyclonal antiserum was capable of detecting IBDV (58,65), and also non-specific presence of IgG complexes in the glomeruli of IBDV infected chickens (74). Utilization of two fluorogenic labels was also demonstrated as a means of quantitation of IBDV replication in cell culture, as a reflection of live/dead cell ratios (5).
The Enzyme Linked Immunosorbent Assay (ELISA) is by far the most widely used diagnostic test in the modern poultry industry, and is also capable of determining the presence of antibodies to many other pathogens. The popularity of commercial ELISA kits results from the ease of performance, accommodation of a large number of samples, high sensitivity, and economical price. Commercial ELISA kits for IBDV quantitate the presence of virus specific antibodies only (109). Variations of the standard ELISA are utilized to determine the presence of virus in IBDV infected tissues, but remain predominantly as a research tool. The Antigen Capture-ELISA developed by Snyder et al. (128) utilizes monoclonal antibodies specific to the surface markers of IBDV to capture virus from tissue homogenates. Field virus populations of IBDV were screened and determined to be relatively homogenous falling into three major antigen types. Antigenic types identified could be segregated into distinct geographical regions of the United States (128). This technology has since been modified to use polyclonal antiserum to capture virus from infected tissues. Early indications are that polyclonal serum may actually bind more virus than the monoclonal antibody, due to the presence of antibodies to multiple antigenic sites (41).
The development of a kinetic based ELISA was utilized to determine the rate of antibody decay. Results from these assays indicated that progeny from parents vaccinated 4 times or more had higher rates of antibody decay than those being vaccinated only twice (129).
Molecular biology procedures have also been refined for the diagnosis, characterization, and identification of classic and variant IBDV. Detection of IBDV in tissue was accomplished utilizing a biotin labeled cDNA derived from segment A at the VP2/VP4 junction. Infected tissues demonstrated specific staining, however, the intensity of the stain did not correlate with virus titer determined by other means (45,57). Randomly derived cDNA probes demonstrated similar results (24). Dot blot hybridization was utilized to detect IBDV in tissue with a probe derived from the cloned sequence of VP1. IBDV could be detected by this method from day 1 to day 24, with the test also demonstrating a wide specificity encompassing both serotypes (58).
The polymerase chain reaction (PCR) has been modified so that it can be utilized for the direct amplification of IBDV from infected tissues (55,142). Because infected samples must first be copied into cDNA by reverse transcriptase, and then amplified with PCR, this procedure is called RT-PCR. A secondary analysis is performed in which restriction enzymes are used to cut the amplified product resulting in the formation of a banding pattern that will identify it as to IBDV serotype classic or variant (55).
Infectious bursal disease virus represents a true paradox, in that it replicates in immune cells and destroys them, yet is capable of stimulating an extremely high immune response (36,52). Because of this immunogenic ability, IBDV infection can be effectively controlled with use of one of any number of vaccination programs. Most IBDV vaccination programs for commercial chickens utilize a combination of a live virus vaccine and inactivated oil emulsion vaccine containing serotype 1 IBDV. Commercial vaccines are available in a number of formulations and concentrations (35,93). The vaccine type employed by the producer will reflect which IBDV is currently causing disease on their premises. Vaccination regimens also vary as to route of administration, timing of administration, number of repetitions, and dose given. Infectious bursal disease virus vaccinations are given to the breeder flock to boost maternal antibody levels that are transferred to the progeny.
Maternal antibody levels are capable of protecting the chick for the first few weeks, but are generally insufficient to protect for the entire growout (29,135). In addition, maternally derived antibody is not uniformly distributed to the progeny, so it is evident that production flocks will contain both susceptible and protected birds at any given time (135).
Earliest vaccinations for IBDV are administered in-ovo at day 18 of embryonation. Virus neutralization (VN) antibody titers in chicks that received the vaccine in-ovo resulted in a resistance to challenge from virulent IBDV up to 10 weeks of age (116). Licensed vaccines are now available for mechanical administration into the yolk sac at 18 days of embryonation (2,38). In general, hatchery vaccination is somewhat questionable because of interference from high levels of maternal antibodies (135). In the hatchery, IBDV vaccination can also be given by microaerosol spray, or subcutaneous injection. Field vaccination for IBDV is customarily delivered in the drinking water.
Infectious bursal disease virus live vaccines are usually derived from attenuated IBDV field virus strains. Attenuation is accomplished by the repeated passage of the virus through cell culture and/or embryonated eggs (126). Live vaccines also spread laterally from bird to bird and carry with them the potential to revert to virulence and, as such, pose a threat to the very birds they were designed to protect (35). There is no evidence at present to indicate that IBDV infection is spread vertically.
Infectious bursal disease virus vaccines are classified by the degree of pathology they induce in the bursa as being mild, intermediate, or invasive. Invasive vaccines induce a high degree of bursal damage which is accompanied by immunosuppression, but produce high antibody titers. Live invasive IBDV vaccines are usually administered in the presence of low levels of maternal antibodies in production birds or following priming with a killed oil emulsion vaccine in breeders (35,76).
Genetically engineered vaccines for IBDV have been described in which various pieces, mainly VP2, are expressed in a number of different vectors. Field testing indicates that most of these products stimulate antibody production, and since they contain only pieces of the IBDV genome, do not pose a threat to virulence. However, antibody produced using these constructs, at best, offer only limited protection and are not economically feasible for widespread application (62,80).
Immunosuppression represents a physiological state in which an individual exhibits temporary or permanent dysfunction of the immune response, resulting from some type of damage to the immune system. Whether temporary or permanent immunosuppression ultimately leads to an increased susceptibility to other disease pathogens. Two basic categories of immunosuppression are considered; one that is antigen specific, and the other is a generalized unresponsiveness of the immune system (119,120).
Antigen specific immunosuppression results in the loss of a specific type, or types, of cell following exposure to the pathogen and can occur directly or indirectly. Direct interference by the pathogen occurs when replication takes place in a specific immune cell or in a cell that in some way plays a role in innate immunity (49). Indirect effects occur when the pathogen interferes with the regulatory cells of the immune system. Both direct and indirect immunosuppression invoke negative consequences for the complete development of antigen specific antibodies. Generalized immunosuppression is most often associated with genetic and/or physiological deficiencies in which the immune cells are absent or the body fails to produce cells or substances necessary to sustain an immune response (49,72.120,121).
Immunosuppression following IBDV infection is characterized by a severe lymphoid cell depletion in the bursa. There is some evidence of additional cellular depletion in the thymus and hematopoietic cell loss in the bone marrow. Lesions induced by IBDV reflect that the virus prefers to replicate in IgM bearing lymphocytes, but can, and will utilize other lymphocytes as well as macrophages throughout the body. Loss of most of the functional B-cells results in the inability to produce antibodies to IBDV or, for that matter, any other pathogen that comes along afterward. Infectious bursal disease virus infection, therefore, has the potential to invoke both direct and indirect effects on the immune system. This, in turn, results in reduced response to vaccines, increased early mortality, and a significantly higher incidence of secondary opportunistic pathogens (105,106).
Immunosuppression induced by IBDV infection is experimentally assessed by the ability or inability of the bird to mount an immune response to IBDV as well as other types of antigens following infection or vaccination. It has been repeatedly demonstrated that vaccination or infection with IBDV at an early age will result in the suppression of the humoral immune response to a number of different antigens (1, 19, 33, 35, 53, 68, 72, 100, 111, 119, 120, 121). In addition, other studies have determined that there is a reduction in natural killer cell activity (115, 117), and reduced immune response to other classes of antigens (102, 118). Challenge with IBDV which is administered to immunologically mature chickens may produce only partial immunosuppression or no immunosuppression at all (28).
Bursectomized birds are still capable of producing an immune response, but because a majority of the immune cells are no longer present, the response is extremely weak. In addition, bursectomized birds do not manifest the physical symptoms of clinical IBDV. The proposed mechanism is that there are insufficient target cells to attain viremia levels to induce lesions. The ability to produce low levels of antibody indicate that immunity in birds to infectious agents can develop, in part, from other lymphoid organs including the spleen, and thymus (36, 95).
Classic and variant serotype 1 IBDV induce different types and intensities of immunosuppression. In-vitro lymphoblast transformation assays demonstrated that the variant IBDV exerted more of an effect on the lymphoid cells, whereas the classic IBDV targeted the humoral immune response (102). Utilization of certain vaccines can also induce immunosuppression, and reflect the need to utilize IBDV vaccines judiciously (35).
Primary pathologic lesions associated with IBDV infection or vaccination of susceptible chickens are found predominantly within the bursa of Fabricius (BF). Rapid onset of lesions within the BF reflect the high concentration of target B-lymphocytes present (120). This is accompanied by the lesser involvement of other lymphoid organs, such as spleen, thymus, cecal tonsils and Peyers patches (64,118). Pathology observed for classic IBDV infection varies from that described for variant IBDV infection. Classic IBDV pathology is well characterized and will be discussed thoroughly in this section with reference to differences demonstrated by variant IBDV (21,107). To summarize, variant IBDV pathology exhibits similar lesions to the classic virus which are less severe, and can be described as highly cytolytic with rapid bursal atrophy and a minimal inflammatory response. One of the proposed mechanisms for this difference in pathology is that variant IBDV, as well as serotype 2 IBDV, induce cytopathology through the process of apoptosis (135,136).
Pathology within the chicken lymphoid system is presumed to result directly from IBDV infection of suitable target cells. Primary target cells have been identified as IgM bearing B lymphocytes, but other cells have also been implicated; they include macrophages. non-B lymphocytes, endothelial cells and reticuloendothelial cells (15,16,48,84,91).
The BF represents a unique organ structure found exclusively in avian species. The bursa is contained within a blind pouch that is located dorsal to the cloaca. This blind pouch is connected by a short duct to the cloaca through which it receives contact with environmental antigens. Exposure to these antigens appears to be a non-specific mechanism at the control of peristaltic movement through the bursal duct due to currents created by transport of waste through the cloaca (105). The BF is divided into distinct follicular regions that can be further divided into a outer dense cortical zone filled with lymphocytes, which surrounds a loosely packed medullary zone of reticuloepithelial cells (97,106). The primary function of the BF is the development, maturation and transformation of the B-lymphocyte, and as a result the bursa strongly influences the humoral immune response of the chicken as well as other avian species (36,67).
There is a direct correlation between the degree of pathological lesions and symptoms induced by IBDV challenge and the number of susceptible cells available for infection (44,51, 66,134). This is perhaps best demonstrated by infection of birds with IBDV following removal of the bursa. Bursectomized birds have little or no antibody production, but show mild lymphocytic necrosis in the spleen and thymus. Presence of the bursa of Fabricius is not necessary to establish infection with IBDV, but it appears to be required for manifestation of the clinical infection (96).
Bursal lesions appear as early as one day following infection and are generally characterized by extensive necrosis of lymphocytes in the cortex of the bursal follicle. Phagocytosis of the necrotic lymphoid cells by the reticular cells then progresses to a severe reticuloepithelial hyperplasia with accompanied inflammation. This is followed by infiltration of heterophils, and accumulation of pyknotic debris within the follicles. As the follicles fill with cellular necrotic debris, the intrafollicular area becomes edematous (105,106). Plasma cells and pyronionphilic blast cells develop in the areas of reticuloepithelial hyperplasia and subsequently become necrotic. Initial atrophy of the bursa is followed by the rapid proliferation of the cortico medullary epithelium and the formation of mucus secreting gland (118). Following IBDV infection, the total bursal tissue surface area can be reduced in excess of 70% with little or no lymphocyte repopulation before age onset involution at approximately 12 weeks of age (108). Secondary lymphoid organs are scattered throughout the avian body. Collectively, these lymphoid tissues have more B-lymphocytes than the bursa, however, they lack the high concentration and as such reactions following IBDV infection go relatively unnoticed (27).
Surveys of tissues from IBDV infected birds utilizing the electron microscope detected morphological changes in the bursa as early as 48 hours. Epithelial microvilli of the bursa were initially reduced in number and size. As the infection progressed, there was gradual involution of the bursal follicles, followed by the development of surface erosions from the loss of epithelial cells (92).
Bursae from IBDV vaccinated birds show marked lymphocyte depletion and infiltration of mononuclear cells similar to what is described for virus challenge (28). However, controlled exposure to live virus vaccination results in overall reduction of the amount of tissue damage. Histological changes were most pronounced in groups of birds vaccinated at 28 days of age. Lesions were characterized by the loss of demarcation between the cortex and medulla, and a significant increase in the number of cells packed into follicles with additional infiltration of mononuclear cells. Depletion of cells from the medulla of the bursa was most severe by 5-7 days post vaccination (35).
Infectious bursal disease virus could be recovered from the intestine from 4-10 days PI for birds challenged at 1, 7, and 14 days. Older challenged birds (5 weeks +) highest intestinal levels were day 3-8 PI. Complement depletion was determined to be significantly lower only in the older birds (5 week +)(29). IBDV antigen can be detected in macrophages and lymphoid cells of the cecum as early as 4 hours PI (84). Other lymphoid organs are adversely affected by the presence of IBDV virus, with pathology attributed to virus specific replication in target cells.
Following IBDV infection the spleen often demonstrates reticuloendothelial cell hyperplasia in the lymphocyte beds that surround the adenoid sheath arteries, with marked splenic fibrinoid degeneration (17, 44, 114). The thymus exhibits a reduction in the cortical mass (19) while in the cecal tonsil there were loss of follicles and a decrease in the number of lymphocytes (17, 44, 114). Kidneys of infected birds demonstrate formation of homogenous casts with infiltration of heterophils and the presence of immunoglobulins (74). Post mortem examination of infected birds often demonstrates infiltration of heterophils and edema in livers associated with hepatic coagulative necrosis. Levels of interferon production increase in the kidney, lungs, thymus, spleen, and bursa following IBDV infection. Attenuated strains of IBDV similar to those utilized for vaccination only induced an increase in interferon levels in the bursa (34).
Other physical manifestations of IBDV infection do not involve the lymphoid system. Secondary pathology induced by IBDV infection is characterized by edema, congestion, hemorrhage, necrosis and heterophilic infiltration in the skeletal muscles of the chest and thigh. In addition, similar lesions have been described (74,95,96), in the intestine (114) (120) and mucosa of the proventriculus (116). Hemorrhage formation, in conjunction with IBDV infection, has also been associated with a disseminated intravascular coagulation defect (122) and the destruction of thrombocytes (138). It can be postulated that IBDV would interfere with immune reactions dependent on multiple immune structures including both the bursa and the thymus (102).
Ivanyi et al. hypothesized that immune complexes may play a role in the pathogenesis of IBDV infections, which could account for the secondary pathologic changes observed (53). Combinations of antigen, antibody and complement represent complexes that contribute to IBDV infection pathology by their physical and pharmacological properties. The lesions of acute immune complex disease typically develop in the vasculature and renal glomeruli resulting in arteritis and glomerulonephritis. Fluorescent antibody staining of infected tissue indicated the presence of gamma globulins in renal glomeruli following infection with IBDV. The presence of these immune complexes within the tissue indicate that they may influence pathogenesis associated with IBDV infection in chickens (74).
Simultaneous multiplication of IBDV and production of antibodies in the bursa may produce immune complexes with cellular damage occurring following the activation of complement, with additional involvement of increased interferon levels. Complement mediated destruction of bursal cells would result in a type of non-inflammatory lymphocyte depletion. Skeeles et al. demonstrated that complement levels increase with age, and associated the increase with an increased severity of lesions induced in older birds following infection with IBDV. It has also been demonstrated that during acute IBDV infection there is a depletion of complement in infected birds on days 3 and 5 PI, with complement levels returning to normal by day 8. Administration of hyperimmune serum during IBDV infection did not appear to increase disease severity (123,124,125).
Apoptosis has also been proposed as one of the mechanisms by which a number of viruses induce pathology. Recent studies have indicated that IBDV also utilizes this mechanism (136,137). Apoptosis is a genetically ordered sequence of events following a very specific cellular signaling stimulus in which irreparably damaged cells are disposed of, with minimal damage to surrounding cells or tissue (133).
Apoptosis should not be confused with necrosis, as each represents a totally different set of events albeit with some overlap. Cellular necrosis results from physical injury and is not in any way genetically controlled. Whereas apoptosis is a genetically predetermined deliberate cellular response to specific developmental and environmental stimuli. Injury events that produce necrosis can trigger apoptosis in response to the presence of cellular debris and inflammation. Likewise, it may be possible that induction of apoptosis could indirectly produce necrosis in some instances where cellular degeneration results from a disease process. Necrosis is typified by the destruction of cytoplasmic organelles and loss of the integrity of the plasma membrane. Apoptosis is associated with the boiling of the cytoplasm, condensation of chromatin, fractures of nuclear DNA and can be distinguished from necrosis by lack of an inflammatory response (139).
Where viruses are concerned, the inhibition of apoptosis has resulted in persistent infections, latency, or enhanced virus production. On the other hand, promotion of apoptosis has been demonstrated to facilitate virus spread and release (133,136,137,139).
Enlargement of the proventriculus has been recognized as a problem in broiler chickens in Northwest Arkansas, as well as in other high intensity poultry producing areas throughout the United States for a number of years. The problem manifests itself at the greatest cost in the processing plant, but may also be associated with poor feed conversion.
Weakened proventriculi that are engorged with feed and digesta are subject to breakage at the point of mechanical evisceration. The contents of the proventriculus contaminate the chicken carcass, resulting in increased levels of washouts, downgrades, and condemnations.
Proventriculitis syndrome has been linked to a number of possible causes. Early considerations involved a longer fasting period before processing. However, increased fasts did not facilitate the emptying of the proventriculus. The search for a plausible explanation of this condition resulted in the determination of a number of factors that adversely affect the function, and structure of the avian proventriculus. A number of dietary components have been associated with enlarged proventriculi. Among them are: biogenic amines, lack of dietary fiber, high levels of mold toxins (37), and copper sulfate supplementation (140). In addition, a number of infectious agents have been identified as exerting an effect on the proventriculus. The pathological infectious agents identified as potential causes of proventriculitis are as follows: reovirus, Marek""s virus, avian influenza, adenovirus (37) and velogenic NewCastle (vNDV) virus. However, there is no evidence, at present, that links any of the preceding factors with the syndrome currently being described.
Bayyari et al. demonstrated that proventriculitis syndrome could be reproduced utilizing an undefined homogenate of affected proventriculi collected from field birds at processing (8). The syndrome could also be reproduced utilizing a filtrate of the same homogenate, and the condition could be exacerbated by the addition of copper sulfate in the feed. Induction of the proventricular syndrome with the filtrate indicated the possibility that the causative agent involved was some type of viral agent. Additional clues indicated that the virus could, possibly be infectious bursal disease virus, as challenged birds seroconverted to IBDV (8, 9).
Further evidence that linked IBDV to this syndrome was found following IBDV challenge after vaccination against IBDV. Unvaccinated challenged control birds exhibited lesions associated with proventriculitis. The following studies were undertaken to elucidate the role of IBDV in the induction of a viral proventriculitis syndrome in chickens. In addition, a number of host and environmental factors will be investigated for the influence they exert on the incidence and severity of this syndrome.
In accordance with the present invention, variant E/1084 infectious bursal disease virus (IBDV) challenge given to specific pathogen free (SPF) chicks at 7, 14, 21, and 28 days post hatch was capable of producing microscopic lesions in the proventriculus, but was only detected in the proventriculus of birds challenged at 28 days post hatch using the AC-ELISA. Birds challenged with Variant E/1084 IBDV on 7, 14 and 21 days post hatch had lower antibody titers at 11 days post challenge than those challenged at 28 days post hatch. Antibody response to Variant E/1084 IBDV on all challenge dates was higher than antibody response to USDA/STC IBDV given at 28 days post hatch. Bursa:body weight (B:BW) ratios were significantly different for birds challenge with Variant E/1084 IBDV on days 7 and 28 post hatch at 4 days post challenge. There was evidence of early onset of bursal atrophy for these two challenge groups. On day 11 post challenge all IBDV infected groups demonstrated significant atrophy of the bursa regardless of age at challenge or challenge virus given. Proventriculus:body weight (P:BW) ratios were significantly different at 4 and 11 days post challenge in birds that received Variant E/1084 IBDV on day 7 post hatch and on day 11 post challenge in birds that received challenge on day 21 post hatch. Internal gross lesions involving the papillae were most evident in birds challenged on days 7, 14, and 28 post hatch with Variant E/1084 IBDV, and also in birds challenged on day 28 post hatch with USDA/STC IBDV.
Specific pathogen free (SPF) white leghorn chickens were challenged at 32 days post hatch with 3 different concentrations of USDA/STC IBDV and 3 different concentrations of Variant E/1084 IBDV. Birds were examined at 2, 3, 4, and 11 days post challenge for the presence of virus and lesions in the proventriculus and bursa. AC-ELISA analysis indicated the presence of infectious bursal disease virus (IBDV) was a factor of both the concentration of virus inoculum given and time post exposure. AC-ELISA analysis of the bursa reflected that the percentage of birds testing positive was a factor of time post virus exposure. Serological analysis indicated that all birds challenged with either strain of IBDV were seropositive at 11 days post challenge, and total antibody titer did not reflect the varied amounts of virus inoculum given. Microscopic lesions in the proventriculus were more pronounced in the groups receiving USDA/STC and occurred as an acute lesion and as a chronic lesion, however, there was no indication of a virus concentration effect. Microscopic lesions in the bursa were present in all IBDV challenge groups at all sampling times.
Thin section electron micrographs revealed the presence of virus like particles within the proventriculus of SPF white leghorn chickens at four days post challenge with IBDV. SPF white leghorn chickens exhibit lesions in the bursa and proventriculus following IBDV challenge. Lesion assessment, both grossly and microscopically, indicates that the USDA/STC IBDV is capable of producing the most severe effect on the proventriculus. Pathology from infectious bursal disease virus infection is exacerbated by the presence of copper sulfate supplementation in the feed. The presence of REO S-1133 virus indicated interference in the immune response or IBDV replication, as well as the number of tissue homogenates testing positive for IBDV at 4 days post challenge. Mortality was significantly increased for birds challenged with USDA/STC IBDV in the presence of copper sulfate and/or REO S-1133 virus. Birds which received dietary copper sulfate supplementation had reduced body weights at 4 days post challenge and at 11 days post challenge.
The presence of apoptotic cells in the proventriculus and bursa at 4 days post challenge with USDA/STC IBDV and Variant E/1084 IBDV was determined using a modified TUNEL fluorescent staining procedure. Specific green fluorescence was found in highest quantity in the bursa, no differences in the intensity of staining could be determined for each IBDV strain. Fluorescence was focused predominantly in the medulla and cortex of infected bursae. Proventricular fluorescent staining was found predominantly in the villi and submucosa. Fluorescent intensity of proventricular sections appeared highest in tissue sections from SPF leghorn chickens challenged with USDA/STC IBDV.
Hemolytic complement levels were decreased at 4 days post challenge in all IBDV challenge groups. Highest level of depletion was in the SPF white leghorn chickens challenged with USDA/STC IBDV. Complement levels were recovered by 6 days post challenge which corresponded with measurable levels of neutralizing antibodies against USDA/STC IBDV. IBD virus was detectable by AC-ELISA at this time also, indicating that all the components needed for the stimulation of complement mediated pathology are present and as such remain as one possible mechanism for IBDV induced tissue damage. Virus neutralizing antibodies appeared first in the SPF white leghorn chickens challenged with Variant E/DEL at 6 days post challenge. All IBDV challenged groups had measurable IgG titers at day 8, but there was a subsequent decrease in titer at day 11.
Broiler chickens challenged with USDA/STC IBDV at 35 days post challenge exhibited gross lesions in the bursa and proventriculus at 4 days post challenge in the presence and absence of copper sulfate. Antigen capture ELISA (AC-ELISA) analysis of tissue homogenates indicates that the USDA/STC IBDV was present at 4 days post challenge and was not influenced by the presence of dietary copper sulfate. Broiler chickens did produce an immune response to IBDV following challenge.
To date submissions for AC-ELISA screening of broiler flocks affected with proventriculitis have encompassed at least 46 facilities from 6 different states. Broiler chicken tissue submissions come from flocks ranging in age from 14 to 35 days post hatch. Virus immunoprecipitation following AC-ELISA screening has resulted in the isolation of at least 5 infectious bursal disease viruses of proventricular origin. Virus isolations are primarily from broiler flocks ranging in age from 22-29 days post hatch.
Inoculation of proventricular origin IBDV isolates into 28 day post hatch SPF white leghorn chickens, revealed the production of lesions associated with infectious bursal disease virus infection. At 11 days post challenge all SPF white leghorn chickens demonstrate IgG antibody titers to IBDV following screening of serum with a commercial IBDV ELISA kit.
RT/PCR-RFLP analysis of new proventricular IBDV isolates indicate that the viruses have a unique restriction enzyme pattern within a 700 bp fragment of the VP2 genomic region. The new isolates appear to share characteristics of both the Delaware variant and a more recent California IBDV isolate from the bursa.